Amblyomma Maculatum Research Articles

Molecular detection of Rickettsia amblyommatis and Rickettsia parkeri in ticks collected from wild pigs in Campeche, Mexico

Tick-borne rickettsioses are caused at least by 15 species of Rickettsia of the Spotted fever group, which represent a major emerging and re-emerging public health problem worldwide. Some of these microorganisms have complex cycles involving the interaction of multiple species of ticks and wild and domestic mammals. Rickettsia infection was investigated in ticks collected from wild pigs at six localities in southeastern Mexico. We collected and tested 196 ticks belonging to four species, including Amblyomma maculatum, Amblyomma mixtum, Amblyomma ovale and Riphicephalus microplus, from 13 of 20 (65%) wild pigs sampled. Overall, Rickettsia DNA was detected in 13.8% of ticks tested (10 ♂ and 17 ♀). Of the 27 Rickettsia-positive ticks, six were A. maculatum, and 21 A. mixtum. Sequencing and phylogenetic analysis of the gltA and ompB genes revealed the presence of Rickettsia parkeri sensu stricto in one female A. maculatum and Rickettsia amblyommatis in five A. maculatum (2 ♂, 3 ♀) and 21 A. mixtum ticks (8 ♂, 13 ♀). The finding of two rickettsial agents in ticks collected from a wild pig population that is regularly captured and kept in captivity or hunted as a source of food raises concern about potential disease transmission to humans and domestic animals. However, more investigations are needed to further understand the ecology of Rickettsia species in free-ranging animals and their implications for human health.

Distribution, Dynamics, and Diversity of Questing Ticks in the Lower Midwest.

We investigated the spatiotemporal patterns of medically important tick species in southcentral Indiana in 2018 and 2019. In recent decades, both Amblyomma americanum (Acari: Ixodidae) (Linnaeus) and Ixodes scapularis (Acari: Ixodidae) (Say) have been expanding their ranges into the Midwest. We report updates to the status of A. americanum, I. scapularis, and Dermacentor variabilis (Acari: Ixodidae) (Say) across 13 counties in southern and south-central Indiana. We found more northerly establishment of A. americanum and more widespread establishment of I. scapularis than previously reported in the literature. We also provide the first report of Amblyomma maculatum (Acari: Ixodidae) (Koch) in Indiana. Using negative binomial regression, we observed significant spatial variation among sampling sites in A. americanum adults and nymphs, D. variabilis adults, and I. scapularis nymphs. Further, we found higher densities of A. americanum nymphs and I. scapularis adults in more forested areas as opposed to less forested, more developed areas. We did not find significant interannual variation in tick densities for any species. More southern sites featured higher densities of A. americanum adults and nymphs, D. variabilis adults, and I. scapularis nymphs than more northern sites. Seasonally, we observed the highest peaks in tick activity in the spring and early summer, but I. scapularis adults were most common in the early spring and fall. Average adult A. americanum and adult D. variabilis densities were positively correlated at our sites. Our results reflect the northward expansion of A. americanum in the Lower Midwest, as well as the increasing geographic prevalence of I. scapularis in the region.

Niche divergence among closely related taxa provides insight on evolutionary patterns of ticks

AbstractAimUnderstanding the degree to which closely related taxa diverge in their niche traits could provide insight on their evolutionary patterns, as well as shed some light on the mechanisms underpinning broad‐scale biogeographic patterns. The evolution of ticks was thought to be driven by hosts. However, recent evidence suggests that tick evolution is more likely to be driven by habitat conditions. The Amblyomma maculatum group of ticks provides a good example to test the former, as its incipient speciation raises the possibility of a very rapid adaptation to slightly different environments.LocationThe Americas.TaxaTicks from the Amblyomma maculatum Koch, 1844 (Acari: Ixodidae) group (A. tigrinum, A. triste s.s., A. maculatum s.s. and two intermediate morphs).MethodsWe addressed the question of whether the differentiation of taxa within this group results from ecological factors, either maintaining a similar ecological niche (conservatism) or by occupying distinct niches (divergence). We analysed the distribution of each tick morphotype with ecological niche models. Next, we explored the question of whether these closely related taxa inhabit environments that are more different or more similar than expected by comparing niche overlap in environmental space.ResultsWe found evidence for niche differentiation, showing that the members of the Amblyomma maculatum group exist in and respond to aspects of different environments, leading to geographical variation.Main conclusionsThe analysis of the ecological niches of the Amblyomma maculatum group of ticks indicates niche conservatism for the pairs A. tigrinum—A. maculatum s.s. and A. triste s.s.—A. maculatum s.s, traditionally associated to allopatric speciation; while incipient niche divergence is suggested for the remaining comparisons. These findings add additional evidence to the study of the evolution of ticks, giving support to the hypothesis of habitat conditions driving the evolution of taxa with no strict host specificity.

Equine attachment site preferences and seasonality of common North American ticks: Amblyomma americanum, Dermacentor albipictus, and Ixodes scapularis

BackgroundTicks are common on horses, but recent publications characterizing equine tick infestations in North America are lacking.MethodsTo further understand attachment site preferences of common ticks of horses, and to document the seasonality of equine tick infestation in northeastern Oklahoma, horses from eight farms were evaluated twice a month over a 1-year period. Each horse was systematically inspected beginning at the head and moving caudally to the tail. Attachment sites of ticks were recorded and all ticks collected were identified to species and stage.ResultsHorses (26 males and 62 females) enrolled in the study ranged in age from 1 to 23 years (mean = 12, 95% CI 11–13). A total of 2731 ticks were collected; 84.1% (74/88) of the horses were infested (median = 3 ticks) at one or more examinations. Five tick species were identified, including Amblyomma americanum (78.2%; 2136/2731), Ixodes scapularis (18.2%; 497/2731), Dermacentor albipictus brown variant (2.6%; 71/2731), Dermacentor variabilis (0.7%; 20/2731), and Amblyomma maculatum (0.3%; 7/231). Most ticks were adults (83.6%; 2282/2731), but immature A. americanum (436/2136; 20.4%), D. albipictus (12/71; 16.9%), and A. maculatum (n = 1) were occasionally recovered. Amblyomma americanum were most often attached to the inguinal area, and I. scapularis and D. albipictus were most commonly found on the chest and axillary region (P < 0.0001). Ticks were found on horses in every month of the year. The largest number of ticks (638/2731; 23.4%) were collected in May (P < 0.0001). Amblyomma americanum, primarily immature, was the only tick recovered in September, I. scapularis and D. albipictus predominated October through February, and both A. americanum and I. scapularis were common in March. In the warmer months, April through August, A. americanum was the most common tick, followed by D. variabilis and A. maculatum.ConclusionsThis research confirms that ticks common on horses in North America have attachment site preferences and that ticks infest horses in Oklahoma throughout the year, including during the winter. Additional research is warranted to fully understand the risk these infestations pose to equine health.Graphical abstract

Detection of Rickettsia Species, and Coxiella-Like and Francisella-Like Endosymbionts in Amblyomma americanum and Amblyomma maculatum from a Shared Field Site in Georgia, United States of America.

Two abundant species of aggressive ticks commonly feed on humans in Georgia: the Gulf Coast tick (Amblyomma maculatum) and the Lone Star tick (A. americanum). A. maculatum is the primary host of Rickettsia parkeri, "Candidatus Rickettsia andeanae," and a Francisella-like endosymbiont (AmacFLE), whereas A. americanum is the primary host for R. amblyommatis, Ehrlichia chaffeensis, E. ewingii, and a Coxiella-like endosymbiont (AamCLE). Horizontal transmission of R. parkeri from A. maculatum to A. americanum by co-feeding has been described, and R. amblyommatis has been found infrequently in A. maculatum ticks. We assessed the prevalence of these agents and whether exchange of tick-associated bacteria is common between A. maculatum and A. americanum collected from the same field site. Unengorged ticks were collected May-August 2014 in west-central Georgia from a 4.14 acre site by flagging and from humans and canines traversing that site. All DNA samples were screened with quantitative PCR assays for the bacteria found in both ticks, and the species of any Rickettsia detected was identified by species-specific TaqMan assays or sequencing of the rickettsial ompA gene. Only R. amblyommatis (15) and AamCLE (39) were detected in 40 A. americanum, while the 74 A. maculatum only contained R. parkeri (30), "Candidatus Rickettsia andeanae" (3), and AmacFLE (74). Neither tick species had either Ehrlichia species. Consequently, we obtained no evidence for the frequent exchange of these tick-borne agents in a natural setting despite high levels of carriage of each agent and the common observance of infestation of both ticks on both dogs and humans at this site. Based on these data, exchange of these Rickettsia, Coxiella, and Francisella agents between A. maculatum and A. americanum appears to be an infrequent event.

Recent advances in understanding tick and rickettsiae interactions.

Ticks are haematophagous arthropods with unique molecular mechanisms for digesting host blood meal while acting as vectors for various pathogens of public health significance. The tick's pharmacologically active saliva plays a fundamental role in modulating the host's immune system for several days to weeks, depending on the tick species. The vector tick has also developed sophisticated molecular mechanisms to serve as a competent vector for pathogens, including the spotted fever group (SFG) rickettsiae. Evidence is still inadequate concerning tick-rickettsiae-host interactions and saliva-assisted transmission of the pathogen to the mammalian host. Rickettsia parkeri, of the SFG rickettsia, can cause a milder version of Rocky Mountain spotted fever known as American Boutonneuse fever. The Gulf Coast tick (Amblyomma maculatum) often transmits this pathogenic rickettsia in the USA. This review discusses the knowledge gap concerning tick-rickettsiae-host interactions by highlighting the SFG rickettsia and the Ammaculatum model system. Filling this knowledge gap will provide a better understanding of the tick-rickettsiae-host interactions in disease causation, which will be crucial for developing effective methods for preventing tick-borne diseases.

Presence of Rickettsia Species in Ticks Collected from Companion Animals in Northeastern Georgia, United States.

Tick-borne diseases are a major threat to both humans and their pets; therefore, it is important to evaluate the prevalence of pathogens carried by ticks on companion animals. In this study, attached and unattached Ixodid ticks were removed from companion animals by a veterinary practice in Hall County, Georgia. DNA was extracted from unengorged adult ticks and each was screened for the presence of Rickettsia spp. by polymerase chain reaction (PCR) and sequenced to determine the species present. Two hundred and four adult hard-bodied ticks were identified to species and Rickettsia spp. were found in 19.6% (n = 38) of the 194 analyzed DNA extracts. Rickettsia montanensis was found in Dermacentor variablis (14.7%; n = 25), Amblyomma maculatum (33.3%; n = 2), and Rhipicephalus sanguineus s.l. ticks (25%; n = 4). One Amblyomma americanum tick contained Rickettsia amblyommatis, while Rickettsia felis was found in one Dermacentor variablis tick, serving as the first report of Rickettsia felis in a tick in this region and within this tick vector. This study suggests that there is a risk of companion animals contracting a species of Rickettsia from a tick bite in northeastern Georgia, indicating a need for more investigation and highlighting the importance of tick prevention on pets.

Niche divergence among members of the Amblyomma maculatum Koch, 1844 (Acari: Ixodidae) group of ticks

Niche divergence among members of the Amblyomma maculatum Koch, 1844 (Acari: Ixodidae) group of ticks

Niche divergence among members of the Amblyomma maculatum Koch, 1844 (Acari: Ixodidae) group of ticks

Niche divergence among members of the Amblyomma maculatum Koch, 1844 (Acari: Ixodidae) group of ticks

Established Population of the Gulf Coast Tick, Amblyomma maculatum (Acari: Ixodidae), Infected with Rickettsia parkeri (Rickettsiales: Rickettsiaceae), in Connecticut.

We identified an established population of the Gulf Coast tick (Amblyomma maculatum Koch) infected with Rickettsia parkeri in Connecticut, representing the northernmost range limit of this medically relevant tick species. Our finding highlights the importance of tick surveillance and public health challenges posed by geographic expansion of tick vectors and their pathogens.

Identification of parasitic arthropods collected from domestic and wild animals in Yucatan, Mexico.

The morphological characterization of ectoparasites from domestic and wild animals is crucial to distinguish those that may be involved in the transmission of zoonotic pathogens. The objectives of this work were to identify parasitic arthropods collected from several hosts in Yucatan State, Mexico and to determine the prevalence of the parasite Trypanosoma cruzi in sylvatic specimens of Triatoma dimidiata. Morphological traits and scanning electron microscopy were used to confirm some parasitic identities. In total, 834 parasitic arthropods of various taxa were identified. Amblyomma americanum, Amblyomma maculatum, Amblyomma parvum, Dermacentor variabilis, and Rhipicephalus sanguineus were identified from dogs, horses, sheep, and deer. The flea Ctenocephalides felis was identified from dogs and the human flea, Pulex irritans was found on horses and sheep. The Neotropical deer ked Lipoptena mazamae was identified from brown brocket deer Mazama pandora. The chewing lice Chelopistes meleagridis, Menacanthus stramineus, Menopon gallinae, Myrsidea sp. and Lipeurus caponis were identified from poultry (Meleagris gallopavo and Gallus gallus). Other chewing lice, Tyranniphilopterus sp., Columbicola columbae, and Physconelloides eurysema were identified from wild birds Pachyramphus aglaiae, Patagioenas flavirostris and Zenaida asiatica, respectively. Trypanosoma cruzi was present in 52.5% of sylvatic adult T. dimidiata. Several of these findings represent new records of ectoparasites for Yucatan and new distribution areas in Mexico. The implications for human and animal health are discussed.

Molecular screening for rickettsial bacteria and piroplasms in ixodid ticks surveyed from white-tailed deer (Odocoileus virginianus) and nilgai antelope (Boselaphus tragocamelus) in southern Texas.

A survey of ixodid ticks parasitizing white-tailed deer (Odocoileus virginianus) and nilgai antelope (Boselaphus tragocamelus) was completed during the 2018–2019 public hunt season on the Laguna Atascosa National Wildlife Refuge (Cameron County, Texas) and the East Foundation's El Sauz Ranch in nearby Willacy County (Texas). Anocenter nitens was the predominant tick species identified with 5% of these ticks collected from nilgai. All life stages were encountered in high numbers on white-tailed deer, indicating that deer may be a primary host in this region. Amblyomma maculatum and Amblyomma inornatum were identified from both hosts, while Ixodes scapularis was encountered only on white-tailed deer. This is the first published record of A. inornatum on nilgai. A subset of ticks was used in PCR assays to detect Rickettsia spp., family Anaplasmataceae, Borrelia spp., and Theileria-Babesia spp. Borrelia spp. were not detected in any of the ticks analyzed. Rickettsia parkeri was detected in three A. maculatum adult ticks from deer, Rickettsia sp. endosymbiont sequences were present in all I. scapularis ticks, and Rickettsia amblyommatis was detected in three A. inornatum adult ticks from deer. Sequence analysis of Anaplasmataceae-positive amplicons from A. nitens and A. maculatum had low percent identity to published Anaplasma spp. sequences, suggesting a unique Anaplasma sp. may be circulating in the population. Anaplasma platys was detected from A. nitens larvae and an Ehrlichia sp. Delta strain was present in A. maculatum, both of unknown pathogenicity towards deer. Theileria cervi was detected in all stages of A. nitens ticks, and positive ticks originated from 27 of 31 deer and a single nilgai sampled from throughout the survey site. The primary vector for T. cervi is absent from this region, suggesting T. cervi is possibly maintained by a different tick species.

&lt;p class="Body"&gt;&lt;strong&gt;Detection of a &lt;em&gt;Rickettsia &lt;/em&gt;sp. and an &lt;em&gt;Ehrlichia chaffeensis&lt;/em&gt;-like organism in ticks parasitizing the endangered Florida Grasshopper Sparrow (&lt;em&gt;Ammodramus savannarum floridanus&lt;/em&gt;)&lt;/strong&gt;&lt;/p&gt;

Between 2013 and 2015, 163 resident endangered Florida Grasshopper Sparrows (Ammodramus savannarum floridanus) and four migratory Eastern Grasshopper Sparrows (A. savannarum pratensis) were examined for the presence of ticks in peninsular Florida. Thirteen Amblyomma maculatum and seven Haemaphysalis chordeilis ticks were removed from 13 Florida Grasshopper Sparrows. Two A. maculatum were discovered on two Eastern Grasshopper Sparrows. Polymerase chain reaction (PCR) and sequencing of resultant amplicons of some of the tick specimens were performed to determine if ticks were infected with pathogenic bacteria. Salivary gland and midgut contents of five of six (83%) of the H. chordeilis tested positive for a novel Rickettsia closely related to, but distinct from, Rickettsia aeschlimannii (causative agent of Mediterranean spotted fever-like illness), an infectious zoonotic bacterium that has not been previously reported in the United States. Four of 14 (29%) of the A. maculatum tested positive for an agent most closely related to an uncultured Ehrlichia previously isolated from Oriental house rats (Rattus tanezumi; 97.5% identity to GenBank KM817187), which is genetically similar to Ehrlichia chaffeensis (causative agent of human monocytic ehrlichiosis), another infectious zoonotic bacterium. Blood from 16 Florida Grasshopper Sparrows and one Eastern Grasshopper Sparrow tested negative for spotted fever group rickettsiae, Anaplasma spp. and Ehrlichia spp. We recommend that additional collections and screening of ticks and blood from Florida Grasshopper Sparrows be undertaken to determine the rates of infection with rickettsiae and ehrlichiae in these imperiled songbirds.

Comparative population genetics of Amblyomma maculatum and Amblyomma americanum in the mid-Atlantic United States

The Gulf Coast tick, Amblyomma maculatum, is undergoing a northward expansion along the United States East Coast, most recently establishing populations in Virginia, Maryland, and Delaware. This expansion has human health implications, as A. maculatum is the primary natural vector of the bacterium Rickettsia parkeri, which causes a spotted fever-type rickettsiosis. Newly established populations of A. maculatum in Virginia tend to have high prevalence of R. parkeri, compared to lower infection rates in the historical range. The factors contributing to high R. parkeri prevalence in Virginia are not known. Investigating connectivity between sites colonized with A. maculatum can help determine whether sites with higher prevalence are isolated or well-connected through migration, thus serving as a source of infected individuals. We characterized 16S rRNA haplotypes of A. maculatum and, for comparison, the congeneric Amblyomma americanum collected from sites where these species co-occur. We then explored connectivity and genetic structure among Virginia populations using pairwise ΦST and AMOVA analyses. Our study identified one recently restored native grassland site with low A. maculatum haplotype diversity and strong evidence of a founder effect, whereas most sites are haplotypically diverse but with no clear genetic structure or connectivity between sites. These findings contrast with high connectivity and a slight mainland/island structure among A. americanum populations. Our results suggest that A. maculatum populations occasionally arise following long-distance drop-offs of few individual ticks in suitable habitat, but no clear migration patterns were observed. The distinct population genetic patterns between species might result from differences in host utilization.

Reproductive incompatibility between Amblyomma maculatum (Acari: Ixodidae) group ticks from two disjunct geographical regions within the USA.

The Amblyomma maculatum Koch group of ixodid ticks consists of three species: A. maculatum, A. triste, and A. tigrinum. However, since Koch described this group in 1844, the systematics of its members has been the subject of ongoing debate. This is especially true of A. maculatum and A. triste; recent molecular analyses reveal insufficient genetic divergence to separate these as distinct species. Further confounding this issue is the discovery in 2014 of A. maculatum group ticks in southern Arizona (AZ), USA, that share morphological characteristics with both A. triste and A. maculatum. To biologically evaluate the identity of A. maculatum group ticks from southern Arizona, we analyzed the reproductive compatibility between specimens of A. maculatum group ticks collected from Georgia (GA), USA, and southern Arizona. Female ticks from both Arizona and Georgia were mated with males from both the Georgia and Arizona Amblyomma populations, creating two homologous and two heterologous F1 cohorts of ticks: GA ♀/GA ♂, AZ ♀/AZ ♂, GA ♀/AZ ♂, and AZ ♀/GA ♂. Each cohort was maintained separately into the F2 generation with F1 females mating only with F1 males from their same cohort. Survival and fecundity parameters were measured for all developmental stages. The observed survival parameters for heterologous cohorts were comparable to those of the homologous cohorts through the F1 generation. However, the F1 heterologous females produced F2 egg clutches that did not hatch, thus indicating that the Arizona and Georgia populations of A. maculatum group ticks tested here represent different biological species.

Rickettsia parkeri (Rickettsiales: Rickettsiaceae) detected in Amblyomma maculatum ticks collected on dogs in Tabasco, Mexico.

The present study was carried out to identify Rickettsia species with zoonotic potential in ticks collected from dogs in a rural area in Tabasco, Mexico. In total 197 Amblyomma maculatum ticks were collected from 40 domestic dogs. The collected specimens were pooled and subjected to DNA extraction. A fragment (380bp) of citrate synthase gene (gltA) was amplified by polymerase chain reaction (PCR) using universal primers for Rickettsia. A second PCR was later performed to amplify a fragment (420bp) of the outer membrane protein B gene (ompB). The PCR products were purified, sequenced and compared using the basic local alignment search tool (BLAST). Twenty out of 40 (50%) tick pools assayed were positive for rickettsial DNA using both primer pairs. The consensus sequence obtained from the ompB gene fragments showed 99.5-100% of identity with strains of Rickettsia parkeri. This study provides the first molecular evidence of the presence of R. parkeri in A. maculatum ticks infesting domestic dogs from southeastern Mexico. Close contact between dogs and humans should lead to consider the infection caused by this species of Rickettsia among the differential diagnoses for people of Tabasco, Mexico, who show acute febrile syndrome associated to inoculation eschar and have a clinical history of tick exposure.

Survey of Rickettsia parkeri and Amblyomma maculatum associated with small mammals in southeastern Virginia

Small mammals are often parasitized by the immature stages of hard-bodied ticks (family Ixodidae) and may serve as reservoir hosts of tick-borne pathogens. Amblyomma maculatum, the Gulf Coast tick, is the primary vector of Rickettsia parkeri, the causative agent of R. parkeri rickettsiosis. This hard-bodied tick species is expanding its historical range from the Gulf Coast of the U.S. up the Mid-Atlantic coast. In Mid-Atlantic states, such as Virginia, R. parkeri prevalence is higher in these ticks than those found in its historical range. This high prevalence may be explained in part by small mammal populations. In this study, small mammals were trapped and checked for the presence of immature A. maculatum. The ticks as well as tissue samples from these mammals were tested for the presence of R. parkeri. This study found six rodent species acting as hosts to immature A. maculatum and three species that may play a role in the enzootic cycle of R. parkeri in Virginia.

Rickettsia parkeri in the Pampa biome of southern Brazil: Isolation, molecular characterization, and serological evidence of canine infection.

In this work we performed a field study in a spotted fever-endemic area of the Pampa biome in Rio Grande do Sul, the southernmost state of Brazil (Rosário do Sul municipality), with the aim to evaluate the serological status of dogs for spotted fever group (SFG) rickettsiae, to attempt the isolation rickettsiae from A. tigrinum ticks, and to further perform a multi-genic molecular characterization of the rickettsial isolate. For this purpose, we collected blood serum samples and ticks from 18 rural dogs during 2016-2017. By immunofluorescence assay, all 18 dogs (100%) reacted serologically to SFG rickettsiae, with highest endpoint titers to R. parkeri, suggesting that at least part of these dogs have been infected by R. parkeri. Among 23 ticks collected from the dogs (13 A. tigrinum and 10 Rhipicephalus sanguineus sensu stricto), 46% (6/13) A. tigrinum contained DNA of R. parkeri, which was successfully isolated in Vero cell culture from one of these ticks. Partial sequences of five genes (gltA, ompA, virB4, dnaA, dnaK) and 3 intergenic spacers (mppA-purC, rrl-rrf-ITS, rpmE-tRNAfMet) were generated from this rickettsial isolate, and were all 100% identical to corresponding sequences of R. parkeri s.s. We provide the first report of a viable isolate of R. parkeri from A. tigrinum, confirming that this isolate belongs to the strain R. parkeri s.s., as it has already been demonstrated for other two tick species of the Amblyomma maculatum species complex (A. maculatum and Amblyomma triste).

Scarcity of Hepatozoon americanum in Gulf Coast tick vectors and potential for cultivating the protozoan.

American canine hepatozoonosis (ACH) is a debilitating tick-borne disease characterized by pyrexia, body wasting, myopathy, mucopurulent ocular discharge, and periosteal proliferation. The causative agent, Hepatozoon americanum, is an apicomplexan that utilizes the Gulf Coast tick, Amblyomma maculatum, as its definitive host and vector. Unlike most tick-borne disease agents, H. americanum is not transmitted via a tick bite, but is transmitted when canids ingest a tick vector that contains sporulated oocysts within the tick hemocoel or paratenic hosts with cystozoites. Our understanding of H. americanum prevalence is based on its detection in the intermediate host, wild or domestic canids, with domestic canids often showing clinical signs at the time of diagnosis. The frequency of H. americanum in A. maculatum, on the other hand, is unknown; this gap in our knowledge hinders our understanding of transmission risk. Furthermore, current diagnostic assays are limited in efficacy, and serologic assays are not widely available. To begin to address gaps in our knowledge, we developed a TaqMan® multiplex qPCR assay for H. americanum detection in A. maculatum tick extracts and evaluated infection rates in questing adult A. maculatum. Additionally, we used a co-culture system to expose H. americanum stages to host cells for in vitro development. Results from qPCR analysis of over 500 tick extracts revealed no positive samples; this suggests both low transmission risk by adult Gulf Coast tick ingestion in the sampled areas, and that surveillance should be focused in areas where ACH has been diagnosed at higher frequencies. Hepatozoon americanum was detectable by qPCR in co-culture of an infected canine buffy coat with ISE6 (Ixodes scapularis embryonic) tick cells, and microscopic examination of samples from those days revealed some structures that were suspicious for developing stages. These data are a starting point for future work to advance our understanding of H. americanum transmission and mechanisms of disease in canids with ACH.

Characterization of the complete mitochondrial genome of Amblyomma ovale, comparative analyses and phylogenetic considerations.

In this study, we sequenced two complete mitochondrial genomes of Amblyomma ovale, a tick of public health importance. Sequencing two distinct individuals, the resulting mitochondrial genomes were 14,756 and 14,760 bp in length and maintained the same gene order previously reported in Amblyomma. These were combined with RNA-seq derived mitochondrial sequences from three additional species, Amblyomma aureolatum, Amblyomma maculatum, and Amblyomma moreliae, to carry out mitogenome comparative and evolutionary analyses against all previously published tick mitochondrial genomes. We described a derivative genome rearrangement that isolates Ixodes from the remaining Ixodidae and consists of both a reverse translocation as well as an event of Tandem Duplication Random Loss. Genetic distance analyses indicated that cox2, nd1, nd5, and 16S are good candidates for future population studies in A. ovale. The phylogenetic analyses corroborated the utility of complete mitochondrial genomes as phylogenetic markers within the group. This study further supplements the genome information available for Amblyomma and facilitates future evolutionary and population genetic studies within the genus.